CN107709975B - Fluorescence detection method and system - Google Patents

Abstract

The present invention relates to multiplex fluorescence detection, including Time Resolved Fluorescence (TRF) detection. The combination of spectral and temporal differences in fluorescence emission and spectral differences in excitation is used to enhance the ability to separate signals from assays for a variety of fluorescent labels. Different types of labels can be used, including up-converting phosphors as well as lanthanide chelates and transition metal chelates. The method may be implemented in an optical plate reader comprising a cartridge-based multi-mode reader.

Description

Fluorescence detection method and system

RELATED APPLICATIONS

This application claims priority to U.S. patent application serial No.14/682,026, filed on 8/4/2015, which is incorporated herein by reference in its entirety.

Technical Field

The present invention relates generally to methods, devices and systems for fluorescence-based detection or measurement of samples, particularly multiplex detection using different fluorophores. The present invention can implement different types of techniques for fluorescence-based detection, including common Fluorescence Detection (FD) and time-resolved fluorescence (TRF) detection.

Background

Protein detection and characterization is an important task in pharmaceutical and clinical researchAnd (5) transaction. Chemiluminescence (CL) is a common method for protein detection in biochemical assays, or for detecting proteins in surface-bound and spatially separated proteins. An example of the latter is the method of sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) in which proteins are electrophoretically transferred to membranes, which is known as the Western Blot (WB) analysis (Towbin et al (1979) Proc. Natl.Acad.Sci.U.S.A.76(9): 4350-. Electrochemiluminescence (ECL) has also been used to detect spot-bound proteins in specially designed multiwell plates (e.g., MULTI)

And MULTl-ARRAY^TMPlate and SECTOR^TMInstrument, Meso Scale Discovery, department of Meso Scale Diagnostics, LLC, Gaithersburg, Md.).

The advantages of CL and ECL are very high sensitivity, with the detection of proteins in solution limited to the sub-picogram/ml range. However, these systems produce transient signals, are not chemically stable, and require complex procedures to produce the chemical reactions required for detection. These systems are also non-linear systems (i.e., one probe generates many photons) and are poorly reproducible and therefore unsuitable for applications requiring quantification of the amount of protein. The last very important limitation is the inability to detect multiple CL signals. The emission of these CL signals is very extensive, which makes the ability to detect two different CL emissions emitted from the same spatial location very challenging.

Fluorescent (FL) probes overcome some of the limitations of CL. Since the relationship between the excitation and emission photons is generally linear, fluorescent probes provide the ability to perform better quantitation. Fluorescent probes are also more versatile because there is no need to contact the probe with other reactive molecules. In general, FL probes are also more stable (especially when shielded from light) because FL probes are typically non-reactive chemicals. Perhaps the most important advantage of FL probes is that they provide the ability to perform multiplex fluorescence detection (multiplexing). FL molecules occur in a variety of forms and have a wide range of excitation and emission bands. Thus, two (or more) probes at the same spatial location can be excited and detected independently, and overlap (or cross-talk) between detection channels is minimized. Color band pass filters are commonly used to report the ability to detect up to four independent fluorophores from the same spatial location. Flow cytometry and multispectral imaging have been reported to have higher levels of multiplex fluorescence detection (Stack et al, (2014) Methods 70(1): 46-58; Perfetto et al, (2004)4(8):648 655).

Unfortunately, FL probes do not exhibit the same sensitivity level as CL and typically have a lower dynamic range. The reason for the lower sensitivity of the FL probe is the presence of background from autofluorescence of the material at the same location or interference from fluorescence of other probes. A different technique, known as time-resolved fluorescence (TRF), was developed in the art to reduce the background from autofluorescence using longer-lived fluorescent probes (Zuchner et al (2009) Anal. chem.81(22): 9449-. In short, autofluorescence generally has a relatively short lifetime (<20ns), so that the TRF detection is delayed in time until the autofluorescence signal disappears. This is technically a time-gated assay, but is commonly referred to as time-resolved (Lakowicz, "Principles of Fluorescence Spectroscopy", 3 rd edition, Springer-Verlag, New York, 2006). The benefits of TRF detection are well documented, including higher sensitivity, lower background and wider dynamic range (Eliseeva & Bunzli (2010) chem. Soc. Rev.39(1): 189-.

Multiple detection of TRF has been reported and has met with some success. The ability to detect two different proteins using Eu and Tb based probes has been demonstrated in biochemical assays using time-resolved fluorescence resonance energy transfer (TR-FRET) (Degorce et al (2009) curr. chem. genomics.3: 22-32; Bookout et al (2000) J. Agric. food chem.48(12): 5868-. In addition, there have been reports of multiplex detection using Eu and Sm, and Eu, Tb and Sm (Bador et al (1987) Clin. chem.33(1): 48-51; Heinonen et al (1997) Clin. chem.43(7): 1142-1150). However, these systems suffer from crosstalk because the emission from one of the lanthanides flows into the detection channel of the other lanthanide. This limits the utility of these methods to having only one truly sensitive channel, the other being limited by the background signal from the second species.

Therefore, there is a need for an improved multiplex system that maintains high sensitivity, background rejection, stability, photobleaching resistance, and dynamic range of time-resolved fluorescence detection with minimal or no cross-talk between channels.

Disclosure of Invention

To address the above problems, in whole or in part, and/or other problems that may have been observed by those skilled in the art, the present disclosure provides methods, processes, systems, devices, apparatuses, and/or devices described by way of example in the embodiments set forth below.

According to an embodiment, a method for multiplex time-resolved fluorescence (TRF) detection comprises: providing a sample comprising a first fluorescent label bound to a first analyte and a second fluorescent label bound to a second analyte, wherein the first fluorescent label has a first fluorescence emission lifetime that is at least 3 times longer than a background fluorescence emission lifetime and has a first excitation wavelength and a first emission wavelength, and the second fluorescent label has a second fluorescence emission lifetime, a second excitation wavelength, and a second emission wavelength; exciting a first fluorescent marker with first excitation light having a first excitation wavelength, wherein the first fluorescent marker emits a first detection signal having a first emission wavelength; exciting a second fluorescent marker with second excitation light having a second excitation wavelength, wherein the second fluorescent marker emits a second detection signal having a second emission wavelength; measuring the intensity of the first detection signal, wherein the intensity of the first detection signal is positively correlated with the amount of the first analyte in the sample; measuring the intensity of a second detection signal, wherein the intensity of the second detection signal is positively correlated with the amount of the second analyte in the sample; wherein the second fluorescence emission lifetime is at least 5 times longer than the first fluorescence emission lifetime.

According to another embodiment, the sample further comprises at least one additional fluorescent label bound to an additional analyte, wherein the additional fluorescent label has a label-specific excitation wavelength, a label-specific emission wavelength, and a label-specific fluorescent emission lifetime that is at least 3 times longer than the background emission lifetime; wherein the first fluorescence emission lifetime, the second fluorescence emission lifetime, and the marker-specific fluorescence emission lifetime are each at least on an order of magnitude different from one another. Exciting the further fluorescent label with label-specific excitation light having a label-specific excitation wavelength, wherein the further fluorescent label emits a label-specific detection signal having a label-specific emission wavelength. The intensity of the label-specific detection signal is then measured, wherein the intensity of the label-specific detection signal is positively correlated with the amount of the additional analyte in the sample. The at least one further fluorescent label bound to a further analyte may also comprise a plurality of different fluorescent labels bound to different analytes.

According to another embodiment, a multi-label time-resolved fluorescence (TRF) detection device or system is configured for performing all or a portion of any of the methods disclosed herein, e.g., the excitation and measurement steps of the method.

According to another embodiment, a device or system for performing fluorescence detection comprises: a processor and a memory configured to perform all or a portion of any of the methods disclosed herein.

According to another embodiment, a computer-readable storage medium comprises instructions for performing all or a portion of any of the methods disclosed herein.

According to another embodiment, an apparatus or system includes a computer-readable storage medium.

According to another embodiment, a multi-labeled Time Resolved Fluorescence (TRF) detection device comprises: a sample support configured to support a sample comprising a first fluorescent marker and a second fluorescent marker, wherein the first fluorescent marker has a first fluorescence emission lifetime that is at least 3 times longer than a background fluorescence emission lifetime and has a first excitation wavelength and a first emission wavelength, and the second fluorescent marker has a second fluorescence emission lifetime, a second excitation wavelength, and a second emission wavelength, and wherein the second fluorescence emission lifetime is at least 5 times longer than the first fluorescence emission lifetime; a light source configured to generate first excitation light of a first excitation wavelength and second excitation light of a second excitation wavelength; a photodetector configured to measure a first detection signal emitted from the sample in response to excitation by the first excitation light and a second detection signal emitted from the sample in response to excitation by the second excitation light; a computing device configured to: controlling the light source according to the time sequence to generate first exciting light and second exciting light; and receiving an electrical output from the photodetector corresponding to the measurement of the first detection signal and the second detection signal.

According to another embodiment, a method for multi-label fluorescence detection comprises: providing a sample comprising a first fluorescent label bound to a first analyte and a second fluorescent label bound to a second analyte, wherein the first fluorescent label comprises an up-converting phosphor (UCP) and the second fluorescent label comprises a non-UCP label; illuminating the first fluorescent label with a first excitation light of a first excitation wavelength, wherein the first fluorescent label emits a first detection signal of a first emission wavelength; illuminating the second fluorescent label with a second excitation light of a second excitation wavelength (which is different from the first excitation wavelength), wherein the second fluorescent label emits a second detection signal of a second emission wavelength (which is different from the first emission wavelength); measuring the intensity of the first detection signal at a first measurement time, wherein the intensity of the first detection signal is correlated with the amount of the first analyte in the sample; stopping irradiating the second fluorescent marker; and measuring an intensity of a second detection signal at a second measurement time different from the first measurement time after ceasing to irradiate the second fluorescent marker, wherein the intensity of the second detection signal is correlated with the amount of the second analyte in the sample.

According to another embodiment, the sample comprises a third fluorescent label bound to a third analyte, the third fluorescent label comprising a non-UCP label different from the second fluorescent label, and the method further comprises: illuminating a third fluorescent marker with a third excitation light of a third excitation wavelength (which is different from the first excitation wavelength and the second excitation wavelength), wherein the third fluorescent marker emits a third detection signal of a third emission wavelength (which is different from the first emission wavelength and the second emission wavelength); stopping the irradiation of the third fluorescent marker; and measuring the intensity of a third detection signal at a third measurement time different from the first measurement time and the second measurement time after stopping the irradiation of the third fluorescent marker.

According to another embodiment, the fluorescence detection device is configured for performing at least the illuminating step and the measuring step in any of the methods disclosed herein, and the fluorescence detection device comprises: a light source configured to generate first excitation light and second excitation light; and a photodetector configured to measure the first detection signal and the second detection signal.

According to another embodiment, the fluorescence detection device comprises: a sample support configured to support a sample comprising a first fluorescent label bound to a first analyte and a second fluorescent label bound to a second analyte; wherein the first fluorescent label comprises an up-converting phosphor (UCP) and the second fluorescent label comprises a non-UCP label; a light source configured to generate first excitation light of a first excitation wavelength and second excitation light of a second excitation wavelength (which is different from the first excitation wavelength); a photodetector configured to measure a first detection signal of a first emission wavelength emitted from the sample in response to excitation by the first excitation light, and a second detection signal of a second emission wavelength emitted from the sample in response to excitation by the second excitation light; and a computing device configured to: controlling the light source to generate first and second excitation lights, respectively, for a predetermined excitation time and for a predetermined time; and controlling the light detector to measure the first detection signal at a first measurement time and to measure the second detection signal at a second measurement time.

According to another embodiment, the sample comprises a third fluorescent label bound to a third analyte, the third fluorescent label comprising a non-UCP label different from the second fluorescent label; the light source is configured to generate third excitation light at a third excitation wavelength (which is different from the first excitation wavelength and the second excitation wavelength); the light detector is configured to measure a third detection signal at a third emission wavelength; and the computing device is configured to: controlling the light source to generate third excitation light for a predetermined excitation time and for a predetermined time; and controlling the light detector to measure a third detection signal at a third measurement time.

According to another embodiment, a fluorescence detection device or system includes a device housing, a cartridge removably mounted in the device housing, excitation optics configured to define an optical path from a light source to a sample, and emission optics configured to define an optical path from the sample to a light detector, wherein: disposing a light source in a cartridge or in a device housing; disposing a light detector in a cartridge or in a device housing; and disposing the computing device in the apparatus housing.

According to another embodiment, a fluorescence detection device or system is configured for performing all or a portion of any of the methods disclosed herein.

According to another embodiment, a device or system for performing fluorescence detection comprises: a processor and a memory configured to perform all or a portion of any of the methods disclosed herein.

According to another embodiment, a computer-readable storage medium comprises instructions for performing all or a portion of any of the methods disclosed herein.

According to another embodiment, an apparatus or system includes a computer-readable storage medium.

Other apparatuses, devices, systems, methods, features, and advantages of the invention will be, or will become, apparent to one with skill in the art upon examination of the following figures and detailed description. It is intended that all such additional systems, methods, features and advantages be included within this description, be within the scope of the invention, and be protected by the accompanying claims.

Drawings

The invention may be better understood with reference to the following drawings. The components in the figures are not necessarily to scale, emphasis instead being placed upon illustrating the principles of the invention. In the drawings, like reference numerals designate corresponding parts throughout the different views.

Fig. 1 is a flow diagram of a method for multi-label time-resolved fluorescence (TRF) detection, according to some embodiments.

Fig. 2 is a schematic diagram of an example of a sample analysis device according to some embodiments.

Fig. 3A is a schematic diagram of an example of a fluorescence detection device according to an embodiment.

Fig. 3B is a schematic diagram of an example of a fluorescence detection device according to another embodiment.

Fig. 3C is a schematic diagram of an example of a fluorescence detection device according to another embodiment.

Fig. 3D is a schematic diagram of an example of a fluorescence detection device according to another embodiment.

Fig. 3E is a schematic diagram of an example of a fluorescence detection device according to another embodiment.

Fig. 3F is a schematic diagram of an example of a fluorescence detection device according to another embodiment.

Fig. 3G is a schematic diagram of an example of a fluorescence detection device according to another embodiment.

Fig. 3H is a schematic diagram of an example of a fluorescence detection device according to another embodiment.

FIG. 4A shows the use of ScanLater^TMSchematic representation of the TRF detection method of the Western Blot detection System (Molecular Devices, LLC, Sunnyvale, Calif.) is shown.

Fig. 4B is a schematic diagram illustrating the principle of TRF detection.

FIG. 5A is an image of a three-fold serial dilution of glutathione S-transferase (GST).

FIG. 5B is a graph of integrated intensity for each band averaging 10 different Western Blots.

FIG. 6 is a diagram showing the use of a camera with ScanLater^TMOf boxes

Graph of stability of Western Blot results over time for reader.

FIG. 7 shows a graph of TRF reagent lack of photobleaching after repeated scanning of a single band on a Western Blot. For each scan, the histogram shows the intensity ("integrated density (int.

Fig. 8 shows Dot Blot results comparing cross-talk emission between detection channels of europium (Eu) and terbium (Tb) based probes.

FIG. 9 shows Dot Blot results comparing cross-talk emissions between detection channels of europium (Eu) and samarium (Sm) -based probes.

FIG. 10 shows the Western Blot results of GST dilution series comparing the cross-talk emission between detection channels of europium (Eu) and ruthenium (Ru) -based probes; these scans are obtained by using a laser diode-excited cell.

FIG. 11 shows a plot of the mean line scan across lanes on the Western Blot shown in FIG. 10; the line scan of the GST Eu-Eu cell is labeled 1101, the line scan of the GST Eu-Ru cell is labeled 1102, the line scan of the GST Ru-Ru cell is labeled 1103, and the line scan of the GST Ru-Eu cell is labeled 1104.

Detailed description of the preferred embodiments

As used herein, the term "analyte" generally refers to the substance to be detected. For example, in other specific embodiments, the first analyte and the second analyte used in the method for performing multiplex TRF detection comprise proteins, more particularly membrane-bound proteins. Analytes may also include antigenic substances, haptens, antibodies, and combinations thereof. Thus, analytes include, but are not limited to, toxins, organic compounds, proteins, peptides, microorganisms, amino acids, nucleic acids, hormones, steroids, vitamins, drugs (including drugs administered for therapeutic purposes as well as drugs administered for illicit purposes), drug intermediates or byproducts, bacteria, virus particles, and metabolites of or antibodies to any of the foregoing.

As used herein, the term "sample" generally refers to a material known or suspected of containing an analyte. Samples obtained from sources may be used directly or may be pretreated to alter the properties of the sample. The sample may be derived from any biological source, such as physiological fluids, including blood, interstitial fluid, saliva, ocular lens fluid, cerebral spinal fluid, sweat, urine, milk, ascites fluid, sputum, synovial fluid, peritoneal fluid, vaginal fluid, amniotic fluid, and the like. The sample may be pre-treated prior to use, for example to prepare plasma from blood, dilute viscous fluids, etc. Methods of pretreatment may include filtration, precipitation, dilution, distillation, concentration, inactivation of interfering components, chromatography, separation steps, and addition of reagents. In addition to physiological fluids, other liquid samples may be used, such as water, food products, etc. for conducting environmental or food production assays. In addition, solid materials known or suspected of containing the analyte may be used as the sample. In some cases, it is advantageous to change the solid sample to a liquid medium or to have the solid sample release the analyte.

As used herein, the term "light" generally refers to electromagnetic radiation that can be quantified as photons. As related to the present disclosure, light may propagate in a wavelength range from Ultraviolet (UV) to Infrared (IR). In the present disclosure, the term "light" is not intended to be limited to electromagnetic radiation in the visible range. In the present disclosure, the terms "light," "photon," and "radiation" are used interchangeably.

The present invention relates to methods for multi-label detection of various types of fluorophores, including long-lived fluorescent dyes and up-converting phosphors (UCPs), by using common Fluorescence Detection (FD), time-resolved fluorescence (TRF) detection, or a combination of both. The ability to utilize a combination of spectral and temporal differences in fluorescence emission for enhanced signal separation in assays from multiple dyes. Also provided are multi-label fluorescence detection devices and systems configured for performing all or a portion of any of the methods disclosed herein, including devices and systems incorporating a cartridge-based optical plate reader (such as a so-called multi-mode reader).

Conventional TRF detection involves exciting a fluorescent marker with a short pulse of light, then typically waiting for a period of time after excitation, and then measuring the remaining long-lived fluorescent signal. In this way, any short-lived fluorescent background signals and scattered excitation radiation are eliminated. This ability to eliminate most of the background signal makes it 2 to 4 orders of magnitude more sensitive than conventional fluorescence. Therefore, TRF detection is designed to reduce background signals from the emission source or from scattering processes (caused by scattering of the excitation radiation) by exploiting the fluorescent properties of certain fluorescent materials.

Typical selection criteria for fluorescent markers for TRF include relatively long emission lifetimes. As mentioned above, it is expected that the marker will signal its well after any brief background signal has dissipated. The long fluorescence lifetime also allows time-gated fluorescence measurements to be made using flash lamp excitation and low cost circuitry. In addition, fluorescent markers may have relatively large "stokes shifts". The term "stokes shift" is generally defined as the shift of a spectral line or band of the luminescent radiation relative to an excitation line or band towards a long emission wavelength. A relatively large stokes shift is desirable because it keeps the excitation wavelength of the fluorescent marker at a large distance from its emission wavelength and because the difference between the excitation wavelength and the emission wavelength is large, it becomes easier to eliminate the excitation radiation from the emission signal. Furthermore, a large stokes shift also minimizes interference from fluorescent molecules in the sample and/or light scattering due to proteins or colloids present with some bodily fluids (e.g., blood). In addition, a large stokes shift also minimizes the requirement for expensive high precision filters to eliminate background interference.

One class of fluorescent compounds that simultaneously have longer emission lifetimes and larger stokes shifts are lanthanide chelates, such as samarium (sm) (iii), dysprosium (dy (iii), europium (eu (iii), and terbium (tb (iii)). Such chelates may exhibit strongly red-shifted, narrow-band, long-lived emission after excitation of the chelate at very short wavelengths. Typically, chelates have strong Ultraviolet (UV) excitation bands due to the chromophore close to the lanthanide in the molecule. After excitation by the chromophore, excitation energy may be transferred from the excited chromophore to the lanthanide. Followed by the fluorescence emission characteristics of the lanthanide. Lanthanide chelates, for example, have exceptionally large stokes shifts, from about 250 nanometers to about 350 nanometers (nm), as compared to fluorescein, which has a stokes shift of only about 28 nanometers. In addition, lanthanide chelates have long fluorescence lifetimes, ranging from about 100 to about 1000 microseconds (μ s), as compared to other fluorescent labels having lifetimes ranging from about 1 to 20 nanoseconds (nm). In addition, these chelates have very narrow emission spectra, typically having bandwidths less than about 10 nanometers at about 50% emission.

Another class of fluorescent compounds having both a longer emission lifetime and a larger Stokes shift are transition metal chelates, such as those of ruthenium (Ru (II)), osmium (Os (II)), and rhenium (Re (I)). The fluorescence lifetime of the transition metal chelate is typically from about 0.1 to about 10 microseconds.

As described above, the present invention relates in one aspect to a novel method of using a multi-labeled long life fluorescent dye for TRF detection. The combination of spectral and temporal differences in fluorescence emission is used to enhance the ability to separate the signal in the assay from multiple dyes. The method takes advantage of the time domain and wavelength domain differences between TRF dyes to reduce crosstalk to less than 1%, and more specifically to less than 0.01%.

The multiplex TRF detection method of the present invention has many advantages compared with conventional methods. For example, improved quantitation can be achieved by using one channel as a reference or standard. For example, when samples are loaded onto a column on a gel for Western Blot using conventional methods, there may be significant errors in how much of the sample actually falls into the lane. By using a reference protein (also known as housekeeping protein) signal in one channel, the signal from the "unknown" protein in the second (or third) channel can then be normalized to the reference channel to improve the relative accuracy.

Another advantage of the multiplex TRF detection method of the invention is that it enables better reference measurements. A common use of Western Blot is to consider phosphorylation of proteins as an indicator of signaling events and calculate the ratio of phosphoprotein to unmodified protein (or total protein). Calculation of such ratios using single-channel Western Blot requires measurement of the first protein, stripping of the Western Blot membrane, and then re-probing and measuring the second protein. The dual channel assay is capable of simultaneously detecting and measuring phosphoproteins and total proteins. This saves a lot of time and improves accuracy because the source of experimental error is eliminated by not having to strip and re-probe.

Fig. 1 is a flow diagram of a method 100 for multiplex TRF detection according to some embodiments. First, a sample is provided comprising a first fluorescent label bound to a first analyte and a second fluorescent label bound to a second analyte, wherein the first fluorescent label has a first fluorescent emission lifetime that is at least three (3) times longer than the background fluorescent emission lifetime and has a first excitation wavelength and a first emission wavelength and the second fluorescent label has a second fluorescent emission lifetime, a second excitation wavelength and a second emission wavelength, and wherein the second fluorescent emission lifetime is at least five (5) times longer than the first fluorescent emission lifetime (step 110). Next, the first fluorescent marker is excited with first excitation light having a first excitation wavelength, wherein the first fluorescent marker emits a first detection signal having a first emission wavelength (step 120). Then, the second fluorescent marker is excited with second excitation light having a second excitation wavelength, wherein the second fluorescent marker emits a second detection signal having a second emission wavelength (step 130). Next, the intensity of the first detection signal is measured, wherein the intensity of the first detection signal is positively correlated with the amount of the first analyte in the sample (step 140). The intensity of the second detection signal is then measured, wherein the intensity of the second detection signal is positively correlated with the amount of the second analyte in the sample (step 150). In particular embodiments, the second fluorescence emission lifetime is at least 100 times or at least 1,000 times longer than the first fluorescence emission lifetime.

In some embodiments, the flow chart of fig. 1 may be considered to schematically illustrate a sample analysis device configured to perform all or a portion of the steps of the method 100 described above. Other implementations of the sample analyzing apparatus are described below.

In other specific embodiments, the method for performing multiplex TRF detection comprises using a second fluorescent label having a second fluorescence emission lifetime in the range of 100 μ s to 1ms, more specifically wherein the second fluorescent label is selected from the group consisting of lanthanide chelates of samarium (sm (iii), dysprosium (dy (iii), europium (eu (iii)), and terbium (tb (iii)). In a further specific embodiment, the method for performing multiplex TRF detection comprises using a first fluorescent label having a first fluorescence emission lifetime in the range of 0.1 to 10 μ s, more specifically wherein the first fluorescent label is selected from the group consisting of transition metal chelates of ruthenium (ru (ii)), osmium (os (ii)), and rhenium (re (i)).

In a further specific embodiment, the stokes shift of the fluorescent label used in a method of performing multiplex TRF detection is greater than about 20 nanometers, in some embodiments greater than about 100 nanometers, and in some embodiments, from about 250 to about 350 nanometers.

In a further embodiment, prior to step 110, the sample is prepared according to the following steps:

a) contacting the sample with:

i) a first antibody that specifically binds to a first analyte;

ii) a second antibody that specifically binds to a second analyte;

iii) a first fluorescent antibody conjugate that specifically binds to the first antibody, wherein the first fluorescent antibody conjugate comprises a first fluorescent label having a first fluorescent emission lifetime, a first excitation wavelength, and a first emission wavelength;

iv) a second fluorescent antibody conjugate that specifically binds to a second antibody, wherein the second fluorescent antibody conjugate comprises a second fluorescent label having a second fluorescence emission lifetime, a second excitation wavelength, and a second emission wavelength; and

b) incubating the sample under conditions and for a time sufficient for the antibody and antibody conjugate to form an immune complex. In some embodiments, the antibody and antibody conjugate may be provided as a mixture in a solution, or the antibody and/or antibody conjugate may be attached to the surface of a solid support. The solid support may be, but is not limited to, a magnetic bead, a gold nanoparticle, a biodegradable organic polymer nanoparticle, a microwell, or a microtiter plate. In other embodiments, the first or second antibody may have the first or second fluorescent label directly attached thereto, thereby eliminating the need for an antibody conjugate. Thus, the disclosed methods include various assays for detecting analytes, such as detecting proteins bound to membranes, proteins bound to beads, proteins in microfluidic channels (potentially separate), proteins in wells of multi-well plates, and/or proteins in gels or other viscous media (potentially separate).

In other specific embodiments, the sample used in the method for performing multiplex TRF detection comprises at least one additional fluorescent label bound to an additional analyte. The additional fluorescent label has a label-specific excitation wavelength, a label-specific emission wavelength, and a label-specific fluorescent emission lifetime, which may be at least 3 times longer than the background emission lifetime. Further, the first fluorescence emission lifetime, the second fluorescence emission lifetime, and the marker-specific fluorescence emission lifetime may each be at least an order of magnitude different from one another. Exciting the further fluorescent label with label-specific excitation light having a label-specific excitation wavelength, whereby the further fluorescent label emits a label-specific detection signal having a label-specific emission wavelength. The intensity of the label-specific detection signal is then measured, wherein the intensity of the label-specific detection signal is positively correlated with the amount of the additional analyte in the sample. The at least one further fluorescent label bound to a further analyte may also comprise a plurality of different fluorescent labels bound to different analytes.

In a further embodiment, in the method for performing multiplex TRF detection, the first analyte is a reference protein and the second analyte is an unknown protein, further wherein the second detection signal is normalized to the first detection signal.

In another embodiment, in the method for performing multiplex TRF detection, the first analyte is a protein and the second analyte is a modified form of the protein, further wherein the ratio of modified protein to unmodified protein is calculated, in particular wherein the modified form of the protein is a phosphorylated form of the protein.

In further embodiments, one or more up-converting phosphors (UCPs) may be used as fluorescent labels, and may be used in combination with other types of fluorescent labels (i.e., "non-UCP" labels), such as lanthanide chelates and transition metal chelates as described herein. As understood by those skilled in the art, UCPs exhibit anti-stokes shifts (or negative stokes shifts) rather than positive stokes shifts. That is, during photon up-conversion, the sequential absorption of two or more photons of excitation light by the UPC causes the UPC to emit light at shorter wavelengths than the excitation wavelength (rather than light at longer wavelengths than the excitation wavelength). In a typical example, a UPC emits emission light in the visible spectrum (e.g., 600nm) in response to absorbing light in the Infrared (IR) spectrum (e.g., 980 nm). Like the non-UCP fluorescent labels described herein, UCP fluorescent labels can be configured (formulated, manufactured) to have a longer emission lifetime. Furthermore, the IR wavelength illuminating the UCP fluorescent marker is significantly different (i.e., spectrally far apart) from the UV wavelength illuminating the non-UCP fluorescent marker, and the background noise is significantly reduced. The emission wavelength of UCP labels is also very different from the emission wavelength of non-UCP labels, providing very good resolution and very low cross-talk in the measurement signal. According to one aspect of the present disclosure, the use of different labels (such as a UCP label and one or more different non-UCP labels) requires the use of different excitation wavelengths, different emission wavelengths, and different emission lifetimes, resulting in very little cross-talk. As with non-UCP labels, the particular excitation wavelength, emission wavelength, and emission lifetime depend on the particular configuration of a given UCP. The use of one or more UCP labels in combination with one or more non-UCP labels (e.g., lanthanide chelates and/or transition metal chelates) in multiplex (e.g., double, triple, etc.) experiments can also reduce the sample experiment time required. In addition, certain UCP labels can be used in common fluorescence-based assays, i.e., detecting/measuring a sample while illuminating the sample (i.e., requiring little or no delay between excitation and detection/measurement of emission), because the background signal intensity associated with UCP is very low. Thus, in some embodiments disclosed herein, methods when using such UCPs in combination with non-UCP markers may require TRF or a combination of normal fluorescence and TRF.

As non-limiting examples, UCPs can be lanthanide-doped inorganic compounds or transition metal-doped inorganic compounds that exhibit anti-stokes shifts. The inorganic compound may be a crystalline material comprising a transparent host lattice doped with one or more dopants capable of or enhancing upconversion activity. Examples of inorganic compounds that form the basis of certain UCPs include, but are not limited to, various halides (e.g., NaYF)₄、YF₃、LaF₃) Oxide (e.g. Y)₂O₃、ZrO₂) And oxysulfides (e.g. Y)₂O₂S、La₂O₂S). Examples of suitable dopants include, but are not limited to, trivalent lanthanide ions and transition metals, such as erbium (Er)³⁺) Thulium (Tm)³⁺) Holmium (Ho)³⁺) Praseodymium (Pr)³⁺) Neodymium (Nd)³⁺) Dysprosium (Dy)³⁺) Ytterbium (Yb)³⁺) And/or samarium (Sm)^{3 +}). As another example, can be as

The UCP is used as described in Terhi, Upconverting Phosphor Technology Exceptional Phosphor Properties Light Up geographic biological Assays, University of Turku Publications (2011), the entire contents of which are incorporated herein by reference. As another example, a suitable UCP may be manufactured by Intelligent Material Solutions Inc., Princeton, N.J., USA

UCP nanocrystals, and can be purchased from Sigma-Aldrich, inc., st.louis, Missouri, USA.

The methods disclosed herein can be carried out using a suitable sample analysis device. Examples of suitable sample analysis devices are described below with reference to fig. 2-3H.

Fig. 2 is a schematic diagram of an example of a sample analysis device 200 according to some embodiments. The sample analysis device 200 is configured to perform multiple fluorescence detections on a sample to detect multiple analytes, where these terms have been defined elsewhere herein. In some embodiments, the sample analysis device 200 is configured to enable a user to select a desired type of optical measurement to be made, which is not only a TRF measurement but also other fluorescence-based measurements, as well as other types of optical measurements, such as, for example, luminescence, absorbance, cell imaging, and the like. For example, a user can reconfigure the optics of sample analysis device 200 to perform a desired type of fluorescence measurement. Thus, in some embodiments, the sample analysis device 200 can be a multimode reader. For example, as a multi-mode reader, the sample analysis device 200 can be reconfigured by having a user select an application-specific cartridge among a plurality of different cartridges available, and then load the selected cartridge into the sample analysis device 200 to create an optical and electronic circuit specific to the desired application. The selected cartridge is connected to the sample analysis device 200, whereby the sample analysis device 200 is suitably configured for performing the selected experiment. The cartridge may contain optics that are specific or optimized for a particular type of application (e.g., multiple TRF detection). Internal optics housed in the cartridge may communicate with external optics housed within the housing of the sample analysis device 200 through an optical port of the housing of the cartridge. Some cartridges may additionally include one or more internal light sources and/or one or more light detectors. Examples of cartridge-based multimode readers are described in U.S. patent nos. 9,188,527 and 8,119,066, which are incorporated herein by reference in their entirety.

In general, the structure and operation of the various components provided in an optical-based sample analysis instrument are understood by those skilled in the art and, therefore, are only briefly described herein to facilitate an understanding of the presently disclosed subject matter. In the embodiment shown, the sample analysis device 200 comprises a sample support 204 configured to support one or more samples to be analyzed; and a light detector 208 configured to receive and measure emitted light 212 emitted from the sample. The sample support 204 (when in an operative position for performing optical measurements on a sample), the light detector 208, and other components shown in fig. 2 may be enclosed in a device housing 206 of the sample analysis device 200. The device housing 206 may include one or more panels, doors, drawers, etc. for loading the sample support 204 (and cartridge, if any), access to the interior region of the sample analysis device 200.

In general, sample support 204 may be one or more containers configured to hold one or more samples during analysis. By way of non-limiting example, the sample support 204 may be a multi-well plate (also referred to as a microtiter plate, microwell plate, or optical plate), one or more cuvettes, a substrate that supports a spot or blot (blot) containing the corresponding sample, or the like. Sample support 204 may be disposed on a sample carrier (or sample support carrier) 210 configured to move sample support 204 along one or more axes. For example, the sample carrier 210 may be a manually actuated, semi-automated, or motorized stage or platform. As indicated by the arrows in fig. 2, the sample carrier 210 may be moved into and out of the device housing 206. A sample or a sample support 204 containing one or more samples can be loaded onto the sample carrier 210 when the sample carrier 210 is in an external position (e.g., when the sample carrier 210 is at least partially outside the device housing 206). Thus, the sample carrier 210 may also be considered a sample support. The sample carrier 210 may then be moved to an interior position where the sample carrier 210 is fully seated in the device housing 206 in order to align the sample with the optical components and/or liquid handling components of the sample analysis device 200 (or to align multiple samples in succession).

In various embodiments, the light input end of the light detector 208 generally includes a lens. The output may include electrical connectors (e.g., contacts, terminals, plugs, wire holders, etc.) to provide power and enable the measurement signals generated by the light detector 208 to be output into signal processing circuitry (e.g., data acquisition circuitry) provided by the sample analysis device 200 or located external to the sample analysis device 200. According to this embodiment, the light detector 208 may be a photomultiplier tube (PMT), photodiode, Charge Coupled Device (CCD), Active Pixel Sensor (APS) such as a Complementary Metal Oxide Semiconductor (CMOS) device, or the like, if needed to optimize the sensitivity of the emission wavelength to be detected.

In typical embodiments, the sample analysis device 200 also includes emission optics 216 configured to transmit the emitted light 212 from the sample to the light detector 208. The emitting optics 216 may also be configured to process the emitted light 212. Examples of processing include, but are not limited to, collection, focusing, alignment, filtering, beam steering, beam splitting, and beam path switching. Thus, in accordance with this embodiment, the emission optics 216 may include one or more lenses, readheads, apertures (apertures), filters, light guides, mirrors, beam splitters, monochromators, diffraction gratings, prisms, optical path switches, and the like. The emission optics 216 may be configured to receive the emitted light 212 from above the sample (e.g., a top read head) and/or below the sample (e.g., a bottom read head).

In some embodiments, the sample analysis device 200 further includes a liquid dispensing system 220 (e.g., a syringe needle, tubing, pump, reservoir, etc.) configured for adding liquid to the sample (e.g., into a selected well, or onto a selected footprint of the sample support 204) before or after the sample has been operatively positioned in the sample analysis device 200. For example, a labeling reagent may be added to the sample for fluorescence, luminescence, or other types of measurements, as will be appreciated by those skilled in the art. In some embodiments, two or more different types of reagents may be added.

In embodiments where excitation is desired (e.g., the fluorescence detection techniques disclosed herein), the sample analysis device 200 includes one or more light sources 224 for generating excitation light 228 of a desired wavelength toward the sample. According to this embodiment, the light source 224 may include a broadband light source (e.g., a flash lamp) or one or more Light Emitting Diodes (LEDs), Laser Diodes (LDs), lasers, or the like. A plurality of light sources 224 may be provided to enable a user to select a desired excitation wavelength. In an exemplary embodiment, the sample analysis device 200 further includes excitation optics 232 configured to transmit excitation light 228 from the light source 224 onto the sample. As described above, the excitation optics 232 may include, for example, one or more lenses, readheads, apertures, filters, light guides, mirrors, beam splitters, monochromators, diffraction gratings, prisms, optical path switches, and the like.

In embodiments where the light source 224 is an LED light source, the sample analysis device 200 (or a cartridge operably connected to the sample analysis device 200) may have an electronic current source capable of pulsing the LED light source, a controller for varying the intensity of the excitation light from the LED light source, and/or a photodiode capable of measuring the intensity of the excitation light generated by the LED light source, which may be used to stabilize the LED light source. Preferred LED light sources are available from Lumileds, (San Jose, ca); luxeon Star, Nichia, (japan, german); and Roithner-Laser (Austria, Vienna). In other embodiments, the light source 224 may be a xenon flash lamp module having a xenon flash lamp as the light source and corresponding electronics for generating the pulsed light beam. In the case of using a broadband light source such as a xenon flash lamp, the optical system includes a wavelength selector, a filter, and the like for controlling the wavelength of the excitation light. Preferred xenon flash lamp modules are available from Excelitas (Waltham, massachusetts) and Hamamatsu Photonics (japan).

As also shown schematically in fig. 2, the sample analysis apparatus 200 may further include a computing device (or system controller) 236. As understood by those skilled in the art, the computing device 236 may represent one or more modules configured for controlling, monitoring and/or timing various functional aspects of the sample analysis apparatus 200, and/or for controlling, monitoring and/or timing various functional aspects of the sample analysis apparatus 200The sample analysis device 200 receives data or other signals (e.g., measurement signals from the light detector 208) and transmits control signals to the light detector 208 and/or other components. For all these purposes, the computing device 236 may communicate with the various components of the sample analysis apparatus 200 via wired or wireless communication links, as shown by the dashed lines between the computing device 236 and the light detector 208. For simplicity, other communication links that may exist between the computing device 236 and other components of the sample analysis apparatus 200 are not shown. In typical embodiments, the computing device 236 includes a main electronic processor that provides overall control, and may include one or more electronic processors configured for dedicated control operations or specific signal processing tasks. The computing device 236 may also include one or more memories and/or databases for storing data and/or software. Computing device 236 may also include computer-readable medium 236 comprising instructions for performing any of the methods disclosed herein. Functional modules of the computing device 236 may include circuitry or other types of hardware (or firmware), software, or both. For example, the module may include signal processing (or data acquisition) circuitry for receiving measurement signals from the light detector 208 and software for processing the measurement signals (e.g., for generating graphical data). Computing device 236 may also represent one or more types of user interface devices, such as user input devices (e.g., keypads, touch screens, mice, etc.), user output devices (e.g., display screens, printers, visual indicators or alarms, audible indicators or alarms, etc.), Graphical User Interfaces (GUIs) controlled by software, and devices for loading media readable by an electronic processor (e.g., logic instructions residing in software, data, etc.). The computing device 236 may include an operating system (e.g., Microsoft Windows (R)) for controlling and managing various functions of the computing device 236

Software).

According to some embodiments, experiments using the analysis device 200 for optical measurements may be performed as follows. The sample is introduced into the sample analysis apparatus 200 and placed in an appropriate operational position relative to the optics and other components of the sample analysis apparatus 200. Typically, the "operating" position of the sample is the "optically aligned" position, i.e., the position that establishes an optical path sufficient to acquire optical data from the sample. Depending on the experiment, the operational position may also correspond to being "in fluid alignment" with the sample and sample analysis device 200, i.e., in a position such that fluid can be dispensed onto the sample, for example, by operating the liquid dispensing system 220. Sample introduction may involve loading one or more samples into one or more wells of a microplate or other type of sample support 204 (e.g., preparing samples according to blotting techniques such as Western Blot, as understood by those skilled in the art), and loading or mounting the sample support 204 in the sample analysis device 200, e.g., using a sample carrier 210 as described above. Also as described above, depending on the sample and the type of sample to be measured, the sample may be prepared or processed (incubated, mixed, homogenized, centrifuged, buffered, added with reagents, analytical separations such as solid phase extraction, chromatography, electrophoresis, etc.) prior to being placed in the sample analysis apparatus 200, as will be appreciated by those skilled in the art.

In addition to sample introduction, the sample analysis device 200 or certain components thereof (optics, electronics, etc.) may need to be configured for carrying out a particular type of measurement to be taken. For example, if the sample analysis device 200 is cartridge-based, a suitable cartridge (or cartridges) may be installed in the sample analysis device 200. After the cartridge is mounted, the optics disposed in the cartridge become part of the optical path within the housing 206 of the sample analysis apparatus. For example, the cartridge optics may be aligned with (in optical communication with) the emission optics 216 and the light detector 208, and in some embodiments, may also be aligned with the excitation optics 232 and the light source 224. The mounting of the cartridge provides for the establishment of electrical paths for the transmission of power, data and control signals to and/or from the cartridge.

The sample is then processed as needed to induce photon emission from the sample, where for fluorescence, it may involve the addition of reagents using the liquid dispensing system 220 and/or illumination/excitation using the light source 224 and associated excitation optics 232. The emission optics 216 collect the emission light 212 from the sample and direct the emission light 212 to the light detector 208. The optical detector 208 converts these optical signals into electrical signals (detector signals or measurement signals) and transmits the electrical signals to signal processing circuitry, which may be provided, for example, by the computing device 236 of the sample analysis apparatus 200 as described above. In the case of multiple samples, the sample support 204 may be moved (e.g., by using the sample carrier 210 as described above) to sequentially align each additional sample with the optics for the experiment, thereby sequentially taking measurements of all samples.

As described above, all or a portion of any of the methods disclosed herein may be performed using the sample analysis device 200. Accordingly, the sample analyzer 200 may also be referred to as a fluorescence detection device. For example, the light source 224 may be operated to illuminate the sample with a first excitation signal having a first excitation wavelength optimized for exciting a first fluorescent marker of the sample and a second excitation signal having a second excitation wavelength optimized for exciting a second fluorescent marker of the sample. The light detector 208 may be operated to measure a first detection signal having a first emission wavelength emitted from the sample in response to excitation by the first excitation signal and a second detection signal having a second emission wavelength emitted from the sample in response to excitation by the second excitation signal. For these purposes, in some embodiments, light source 224 may include at least two separate light sources and/or light detector 208 may include at least two separate light detectors.

According to another embodiment, a device for multiplex fluorescence detection (or multiplex fluorescence detection device) is provided. Referring now to fig. 3A, an apparatus 300 for multiplex fluorescence detection is shown. The sample 332 may be held in the device 300 on a sample support 334, such as a microplate or a membrane or substrate, that supports the sample. The device 300 includes a first light source 310 that generates first excitation light 320 and a second light source 312 that generates second excitation light 322. As described above in connection with fig. 2, the device 300 has a first excitation light optical system 324 and a second excitation light optical system 326 with components for directing the first excitation light 320 and the second excitation light 322, respectively, to the sample 332. The sample 332 comprising the first analyte 330 and the second analyte 331 emits a first emission light 340 and a second emission light 342. The device 300 has a first emission light optical system 344 that receives the first emission light 340 and a second emission light optical system 346 that receives the second emission light 342. The first emission light optical system 344 then directs the first emission light 340 to a first detector 350 and the second emission light optical system 346 directs the second emission light 342 to a second detector 352. The above-described components may be located in a main device housing (e.g., device housing 206 shown in fig. 2) of device 300.

Fig. 3B through 3H are schematic diagrams of the components of the device 300 shown in fig. 3A, wherein various components of the device 300 are shown as being contained inside or outside of one or more cartridges, according to some embodiments. That is, such a cartridge may generally include a cartridge housing that encloses (houses) one or more components of the device 300. Such cartridges may be loaded or installed in the device 300 such that the cartridge(s) are enclosed inside a main device housing of the device 300 (e.g., the device housing 206 shown in fig. 2). Examples of cartridge-based readers are described in the above-referenced U.S. patent nos. 9,188,527 and 8,119,066.

Referring now to FIG. 3B, the device 300 further includes a cartridge 360 that includes a first excitation light optical system 324, a second excitation light optical system 326, a first emission light optical system 344, and a second emission light optical system 346.

Referring now to FIG. 3C, the device 300 further includes a cartridge 360 that includes the first light source 310, the second light source 312, the first excitation light optical system 324, and the second excitation light optical system 326.

Referring now to fig. 3D, the device 300 further includes a cartridge 360 that includes a first emission light optical system 344, a second emission light optical system 346, a first detector 350, and a second detector 352.

Referring now to FIG. 3E, the device 300 further includes a cartridge 360 that includes the first excitation light optical system 324, the first light source 310, the first emission light optical system 344, and the first detector 350.

Referring now to FIG. 3F, the device 300 further includes a cartridge 360 that includes the first light source 310, the second light source 312, the first excitation light optical system 324, the second excitation light optical system 326, the first emission light optical system 344, the second emission light optical system 346, the first detector 350, and the second detector 352.

Referring now to fig. 3G, device 300 further includes a first cartridge 360 that includes first light source 310, first excitation light optics 324, first emission light optics 344, and first light detector 350. The device 300 further comprises a second cartridge 370 comprising the second light source 312, the second excitation light optical system 326, the second emission light optical system 346, and the second light detector 352. In alternative embodiments, the first cartridge 360 may not contain the first light source 310 and the first light detector 350 as shown, and in further embodiments, the first light source 310 and the first light detector 350 may both be external to the first cartridge 360. Likewise, the second cartridge 370 may not contain the second light source 312 and the second light detector 352 as shown, and in further embodiments, the second light source 312 and the second light detector 352 may both be external to the second cartridge 370.

It will be appreciated by those skilled in the art that for fluorescence detection methods involving at least one additional fluorescent label bound to an additional analyte, the device and system will be configured for excitation and detection of the additional fluorescent label. For example, an additional light source configured to generate label-specific excitation light at a label-specific excitation wavelength, and an additional light detector configured to measure a label-specific detection signal emitted from the sample in response to excitation by the label-specific excitation light may be used. Where the at least one further fluorescent label bound to a further analyte comprises a plurality of different fluorescent labels bound to different analytes, a plurality of different further light sources and light detectors may be used.

As an example, in the embodiment shown in fig. 3H, the device 300 may further comprise a third light source 313 configured to generate third excitation light 327 in addition to the first and second light sources 310, 312 configured to generate the first and second excitation light 320, 322, respectively. In addition to the first and second light detectors 350, 352 configured to receive and measure the first detection signal 340 emitted from the first analyte 330 and the second detection signal 342 emitted from the second analyte 331, respectively, the apparatus 300 may further comprise a third light detector 353 configured to receive and measure a third detection signal 343 of the third analyte 333 emitted from the sample 332. In addition to the first excitation optics 324 and the second excitation optics 326, the device 300 may also include third excitation optics 327. In addition to the first and second emission optics 344, 346, the apparatus 300 may also include third emission optics 347.

As in other embodiments described herein, one or more of the foregoing components may be provided in one or more cartridges (not shown) that are removably mounted in the device 300. As in other embodiments described herein, the device 300 shown in fig. 3H can be used for dual measurements that employ two different fluorescent labels bound to different analytes. In addition, the device 300 shown in FIG. 3H can be used for triple measurements, which employ three different fluorescent labels bound to different analytes. In other embodiments, the apparatus 300 may include more than three light sources and/or more than three light detectors.

As in other embodiments described herein, the apparatus 200 may further include a computing device, such as the computing device (or system controller) 236 described above and shown schematically in fig. 2, configured to control, monitor and/or time various functional aspects of the apparatus 300, receive data or other signals from the apparatus 300 (e.g., detection signals from the light detector), and send control signals to the light detector and/or other components. For example, in a double experiment, the computing device may be configured to control the first light source 310 and the second light source 312 to generate the first excitation light 320 and the second excitation light 322, respectively, at a predetermined excitation time and for a predetermined time, control the first light detector 350 to measure the first detection signal 340 at a first measurement time, and control the second light detector 352 to measure the second detection signal 342 at a second measurement time different from the first measurement time. The computing device may also be configured to receive an electrical output from each of the light detectors 350 and 352 that corresponds to a measurement of the first detection signal 340 and the second detection signal 342 and correlate the measurement with the amount of the first analyte 330 in the sample 332 and the amount of the second analyte 331 in the sample 332. The computing device may also be configured to control the second light source 312 to stop generating the second excitation light 322 and to control the second light detector 352 to measure the second detection signal 342 after stopping generating the second excitation light 322.

As another example, in a triple experiment, the computing apparatus may be configured to control the third light source 313 to generate the third excitation light 323 at a predetermined excitation time for a predetermined time and to control the third light detector 353 to measure the third detection signal 343 at a third measurement time different from the first measurement time and the second measurement time. The computing device may also be configured for controlling the third light source 313 to stop generating the third excitation light 323 and controlling the third light detector 353 to measure the third detection signal 343 at a third measurement time different from the first measurement time and the second measurement time after stopping generating the third excitation light 323. The computing device may also be configured to receive an electrical output from the third light detector 353 (the electrical output corresponding to a measurement of the third detection signal 343) and correlate the measurement with the amount of the third analyte 333 in the sample 332.

In any of the embodiments shown in fig. 2-3H, certain components of the apparatus 300 may be shared by more than one channel. Therefore, the number of light sources provided may be different from the number of photodetectors provided, or the number of excitation optical device groups or emission optical device groups, or the like. For example, the light detector may have a range of wavelength sensitivities that enable it to effectively detect signals transmitted in two or more channels. As another example, the emission optics may include an emission filter having a wavelength bandpass effective to filter the detection signals transmitted in the two or more channels. Providing common or shared components may reduce the total number of components required for device 300 and the total space required, and may make device 300 and/or the cassette used with device 300 more compact.

An example of a method for multiplex fluorescence detection according to a representative embodiment will now be described. Referring primarily to FIG. 3H, as an example illustrating a fluorescence detection device 300 that may be used to implement the method, it should be understood that other devices or systems described herein may also be configured to implement the method.

According to the method, a sample 332 to be analyzed is provided. The sample 332 can include a first fluorescent label bound to the first analyte 330 and a second fluorescent label bound to the second analyte 331. The first fluorescent label may be or include an up-converting phosphor (UCP) label and the second fluorescent label may be or include a non-UCP label. As described herein, the UCP label may be or include a lanthanide-doped inorganic compound or a transition metal-doped inorganic compound exhibiting an anti-stokes shift; and the non-UCP label may be a transition metal chelate or lanthanide chelate exhibiting a (positive) stokes, or the non-UCP label includes a transition metal chelate or lanthanide chelate exhibiting a (positive) stokes. By way of non-limiting example, the stokes shift of a non-UCP label may be greater than 20nm, or greater than 100nm, or greater than 250nm, or in the range of about 250nm to about 350 nm.

In some embodiments and as described elsewhere herein, sample 332 can be provided by contacting sample 332 with: a first antibody that specifically binds to first analyte 330; a second antibody that specifically binds to the second analyte 331; a first fluorescent antibody conjugate that specifically binds to a first antibody, the first fluorescent antibody conjugate being or comprising a first (UCP) fluorescent label; and a second fluorescent antibody conjugate specifically bound to a second antibody, the second fluorescent antibody conjugate being or comprising a second (non-UCP) fluorescent label. The sample 332 may then be incubated under conditions and for a sufficient time to allow the antibody and antibody conjugate to form an immune complex.

In other embodiments and as described elsewhere herein, the sample 332 may be provided by contacting the sample 332 with a first antibody that specifically binds to a first analyte and a second antibody that specifically binds to a second analyte. The first fluorescent label may be directly attached to the first antibody and/or the second fluorescent label may be directly attached to the second antibody.

The first fluorescent marker is illuminated with first excitation light 320 of a first excitation wavelength. In response, the first fluorescent label emits a first detection signal 340 at a first emission wavelength. The second fluorescent marker is illuminated with second excitation light 322 of a second excitation wavelength (which is different from the first excitation wavelength). In response, the second fluorescent label emits a second detection signal 342 at a second emission wavelength (which is different from the first emission wavelength). In this embodiment, the first excitation wavelength may be in the near infrared range, and the first emission wavelength may be in the visible range. The second excitation wavelength is in the ultraviolet range and the second emission wavelength is longer than the second excitation wavelength. The irradiation of the first fluorescent marker and the irradiation of the second fluorescent marker may be performed simultaneously or sequentially.

The intensity of the first detection signal 340 is measured at a first measurement time. The illumination of the second fluorescent marker is stopped, after which the intensity of the second detection signal 342 is measured at a second measurement time, which may be different from the first measurement time. The intensity of the first detection signal 340 is related to the amount of the first analyte 330 in the sample 332 and the intensity of the second detection signal 342 is related to the amount of the second analyte in the sample 332. Emission optics 344, 346, and 347 may be configured to direct first detection signal 340 and second detection signal 342 through a common emission filter or different emission filters.

In some embodiments and as described elsewhere herein, UCP labels (whether or not they have extended emission lifetimes) are suitable for reading (measurement) according to common fluorescence techniques. In this case, the intensity of the first detection signal 340 may be measured while irradiating the first fluorescent marker. In other embodiments and as described elsewhere herein, UCP tags have extended emission lifetimes, which can be exploited by reading according to TRF techniques. In this case, the irradiation of the first fluorescent marker is stopped, and then the intensity of the first detection signal is measured. In either case, the first measurement time and the second measurement time may be different from each other. Further, the first fluorescence emission lifetime (of the UCP label) and the second fluorescence emission lifetime (of the non-UCP label) may be different from each other. As non-limiting examples, the second fluorescence emission lifetime may be in the range of 0.1 μ s to 10 μ s, or in the range of 100 μ s to 1 ms.

In some embodiments and as described elsewhere herein, the first and second analytes 330, 331 are or include proteins or membrane-bound proteins. In some embodiments, one of analytes 330 and 331 is a reference protein and the other is an unknown protein. In this case, the second detection signal 342 may be normalized to the first detection signal 340 or the first detection signal 340 may be normalized to the second detection signal 342 depending on which analyte 330 and 331 is the reference protein. In further embodiments, one of analytes 330 and 331 is an unmodified protein and the other is a modified or phosphorylated form of the protein. In this case, the ratio of modified or phosphorylated form of protein to unmodified protein may be calculated based on the measured intensities of the first detection signal 340 and the second detection signal 342.

In some embodiments and as described elsewhere herein, multiple samples are provided, for example, in separate wells of a multi-well plate, or in separate blots of a membrane or other suitable substrate. Multiplex fluorescence detection as described herein can be performed on each sample by performing the following steps on each sample: a step of irradiating the first fluorescent marker, a step of irradiating the second fluorescent marker, a step of measuring the intensity of the first detection signal 340, and a step of measuring the intensity of the second detection signal 342. The individual samples may be optically aligned (typically sequentially) with the first light source 310 and the first light detector 350, and with the second light source 312 and the second light detector 352. Optically aligning the sample with the selected light source and light detector may involve optically aligning the sample with a cartridge removably mounted in the device housing of the fluorescence detection device 300 such that the cartridge is in optical and/or electrical communication with the fluorescence detection device 300. As described elsewhere herein, the cassette may enclose one or more components of the fluorescence detection device 300.

In other embodiments, the methods can be extended to the use of three or more different fluorescent labels, and combinations of two or different classes or sets of fluorescent labels (e.g., UCP, lanthanide chelates, transition metal chelates, etc.) can be utilized, which provides the advantages of significantly reducing signal channel crosstalk, etc., as described herein.

As an example of a method of triple fluorescence detection, sample 332 includes a third fluorescent label bound to a third analyte 333. According to this embodiment, antibody and antibody conjugates or direct binding may be performed. As described herein, the third analyte 333 can be, for example, a protein, a membrane-bound protein, a reference protein, an unknown protein, an unmodified protein, or a modified or phosphorylated form of a protein. The third fluorescent label may be a non-UCP label different from the second fluorescent label, or may comprise a non-UCP label different from the second fluorescent label. For example, the second fluorescent label may be or may include a transition metal chelate, and the third fluorescent label may be or may include a lanthanide chelate.

In the triple method described herein, the third fluorescent marker is irradiated with third excitation light 323 of a third excitation wavelength (which is different from the first and second excitation wavelengths). In response, the third fluorescent label emits a third detection signal 343 at a third emission wavelength (which is different from the first emission wavelength and the second emission wavelength). The irradiation of the third fluorescent label is then stopped, after which the intensity of the third detection signal 343 is measured at a third measurement time, which may be different from the first measurement time and the second measurement time. The measurement of the UCP-containing analyte (first analyte 330) can be obtained by ordinary fluorescence or TRF as described above.

In some embodiments, the first excitation wavelength is in the near infrared range, the first emission wavelength is in the visible light range, and at least one of the second excitation wavelength and the third excitation wavelength is in the ultraviolet range. In some embodiments, the UCP of the first fluorescent label has a first fluorescence emission lifetime, the second fluorescent label has a second fluorescence emission lifetime, the third fluorescent label has a third fluorescence emission lifetime, and the first fluorescence emission lifetime is different from the second fluorescence emission lifetime and the third fluorescence emission lifetime. In some embodiments, the second fluorescence emission lifetime is at least 5-fold, or at least 50-fold, or at least 100-fold, or at least 500-fold, or 1000-fold longer than the third fluorescence emission lifetime.

Examples

Protein detection and characterization is an important task for pharmaceutical and clinical research. For example, protein detection and characterization may provide information about up-and down-regulation of proteins in cells, phosphorylation during cell signaling, and expression of transfected proteins. Various techniques have been developed for protein analysis including plate reader-based enzyme-linked immunosorbent assays (ELISA), sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and spot or bead based capture systems using luminescence readings. However, improvements in analytical methods for detecting and quantifying proteins are important to provide better tools to help understand disease mechanisms.

A number of luminescent probes have been developed to detect and analyze proteins. The probes are typically linked to a primary or secondary antibody that then selectively binds the protein of interest. These probes may be fluorescent molecules that produce light when excited with electromagnetic radiation, or reactive species that produce light when contacted with another reactive molecule (e.g., a substrate) or some other stimulus (e.g., an electrical current). Such probes are versatile in that they can be attached to proteins, nucleotides or small molecules by well-known chemical reactions. The relative amount of protein in the sample can be determined by the amount of light produced by the probe, thereby enabling quantification of the protein. Such probes also facilitate the determination of the spatial location of the protein of interest from low resolution (100-. These probes may be organic dyes, inorganic compounds, fluorescent proteins or enzymes.

Chemiluminescence (CL) is a common method for protein detection in biochemical assays, or for surface-bound and spatially-separated protein detection. An example of the latter is the method of sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) in which proteins are electrophoretically transferred to membranes, which is known as the Western Blot (WB) analysis (Towbin et al (1979) (1979) Proc. Natl. Acad. Sci. U.S.A.76(9): 4350-. Electrochemiluminescence (ECL) has also been used to detect spot-bound proteins in specially designed multiwell plates (e.g., MULTI)

And MULTl-ARRAY^TMPlate and SECTOR^TMInstrument, Meso Scale Discovery, department of Meso Scale Diagnostics, LLC, Gaithersburg, Md.).

The advantages of CL and ECL are very high sensitivity, with the detection of proteins in solution limited to the sub-picogram/ml range. However, these systems produce transient signals, the systems are unstable, and require complex procedures to produce the chemical reactions required for detection. These systems are also non-linear systems (i.e., one probe produces many photons) and are poorly reproducible and therefore unsuitable for applications requiring quantification of the amount of protein. Finally, an important limitation is the inability to multiplex the determination of multiple CL signals. Their emissions are very broad, which makes the ability to detect two different CL emissions from the same spatial location very challenging.

Fluorescent (FL) probes overcome some of the limitations of CL. Fluorescent probes provide better quantification capability since the relationship between the excitation and emission photons is generally linear. Fluorescent probes are also more versatile because there is no need to contact the probe with other reactive molecules. In general, FL probes are also more stable (especially when shielded from light) because FL probes are typically non-reactive chemicals. Perhaps the most important advantage of FL probes is that they provide the ability to perform multiple assays. FL molecules occur in a variety of forms and have a wide range of excitation and emission bands. Thus, two (or more) probes located at the same spatial position can be excited and detected independently, and overlap (or cross-talk) between the detection channels is minimized. The ability to detect up to four independent fluorophores from the same spatial location by using color band pass filters has been reported many times. Flow cytometry and multispectral imaging have been reported to have higher levels of multiplex detection (Stack et al, (2014) Methods 70(1): 46-58; Perfetto et al, (2004)4(8):648 655).

Unfortunately, FL probes do not exhibit the same sensitivity level as CL and generally have a lower dynamic range. The reason for the lower sensitivity of the FL probe is the presence of background from autofluorescence of the co-localized material or fluorescence interference from other probes. A different technique, known as time-resolved fluorescence (TRF), was developed in the art to reduce the background from autofluorescence using longer-lived fluorescent probes (Zuchner et al (2009) Anal. chem.81(22): 9449-. In short, autofluorescence generally has a relatively short lifetime (<20ns), so that the TRF detection is delayed in time until the autofluorescence signal disappears. This is technically a time-gated assay, but is commonly referred to as time-resolved (Lakowicz, "Principles of Fluorescence Spectroscopy", 3 rd edition, Springer-Verlag, New York, 2006). The benefits of TRF detection are well documented, including higher sensitivity, lower background and wider dynamic range (Eliseeva & Bunzli (2010) chem. Soc. Rev.39(1): 189-.

A great deal of effort has been made to exploit the most prevalent entities based on Eu and Tb to develop and optimize a TRF probe based on a lanthanide coordination complex (Kemper et al (1999) J.Biomol. Screen.4(6): 309-. In addition to membranes, these probes have other broad uses and show good sensitivity for detecting proteins in tissue sections and living cells (Su et al (2005) anal. biochem.347(1): 89-93; Gahlaut & Miller (2010) Cytometry A. Dec2010; 77(12): 1113-1125). Various instruments have been developed to measure TRF, particularly two-dimensional arrays. Imaging of lanthanide probes can be performed using standard camera systems and Ultraviolet (UV) excitation, although sensitivity is reported only in the nanogram range (Kemper et al (2001) electrophosphoresis.22 (5):881- > 889). Sensitivity can be improved by using time-gated cameras with short pulses or pulsed high-intensity UV light sources (Gahlaut & Miller (2010) Cytometry A.77(12): 1113-. However, this can increase the overall cost of the instrument. Point scanning systems have been developed using pulsed UV lasers and time-gated photon counting (Zuchner et al (2009) anal. chem.81(22): 9449-. Doto Blot and Western Blot are reported to have superior sensitivity and extended dynamic range compared to chemiluminescence and fluorescence.

We have demonstrated the ability to detect and quantify membrane-bound proteins labeled with TRF staining. The membrane is incubated with a europium chelate-labeled secondary antibody or streptavidin that specifically binds to the protein of interest. Europium (Eu) has a long fluorescence lifetime (approximately 1 msec) and exhibits a delay time of 50 μ sDetection in fluorescence from resolution (TRF) mode can significantly reduce background or other short-lived radioactive sources from autofluorescence (fig. 4A and 4B). FIG. 4A shows the use of ScanLater^TMSchematic representation of one non-limiting example of a TRF detection method of the Western Blot detection System (Molecular Devices, LLC, Sunnyvale, Calif.), which in some embodiments may utilize cassettes configured specifically for WB detection. The existing primary antibody 401 binds to the protein of interest 402. Then, Eu-labeled secondary antibody 403 is bound to primary antibody 401. Then ScanLater is added^TMThe system is used to detect (measure) 404. It will be appreciated that other than scanlatex may be used^TMOther detection systems than systems. Fig. 4B is a schematic diagram showing the principle of TRF detection. Fig. 4B plots the intensity of the lamp excitation pulse and the decay of the fluorescence as a function of time, where time 0 corresponds to the start of the excitation pulse. Fig. 4B also shows the time period during which the previous excitation pulse can be measured.

The film was placed in a flatbed reader system where it was scanned using a flash lamp-based TRF cassette that had been optimized for WB scans. The flash lamp reduces the cost of the system compared to previously reported pulsed ultraviolet laser systems while maintaining sensitivity (Zuchner et al (2009), anal. chem.81(22): 9449-. The method does not involve enzymatic detection and the Eu-chelate is resistant to photo-bleaching, so the signal remains stable for a long time (weeks to months). This enables repeated readings of the membrane and the possibility of comparing the band intensities with known standards to obtain more accurate quantitative results.

TRF detection adopts photon counting; the theoretical dynamic range is therefore>10⁵. In fact, the dynamic range is limited by saturation of binding sites on the high abundance bands and non-specific binding to background membranes. There is no camera "flashing" in the saturation of high intensity light as occurs with chemiluminescence or fluorescence detection, so the system has a clear band and excellent image quality. The system provides membrane-bound protein analysis with high sensitivity, wide dynamic range and long-term stabilitySubstrate-free environment. The advantage of this system over existing systems is that it can improve quantification and can rescan samples for reference or as an instrument standard.

Three-fold serial dilutions of glutathione S-transferase (GST) were used to demonstrate

ScanLater from Multimode Detection Platform (Molecular Devices, LLC, Sunnyvale, Calif.) scanning^TMDynamic range of (fig. 5A). For detection of the GST protein, a biotin-labeled rabbit anti-GST primary antibody was used. Using ScanLater^TMAnd detecting Eu-labeled streptavidin. The blot was washed, dried and scanned. The system showed a subpicogram detection limit for GST with more than 4 log positive response of the signal to the amount of GST (see fig. 5B). Fig. 5A is an image of a GST dilution series, and fig. 5B is a plot of integrated intensity of a single band from an average of 10 different western blots.

Limitations of CL and FL detection methods include signal stability. In the case of typical CL reagents, the signal stabilizes for 5 to 20 minutes, after which the substrate is used up and the band intensity decreases. For FL, the organic fluorescent agent is more stable when kept under the proper conditions, but the organic fluorescent agent is susceptible to photo-bleaching, and the signal will decay after repeated exposure to excitation light. TRF detection avoids these two limitations and provides improved stability performance. To show long-term stability, three-fold serial dilutions of GST were used to demonstrate signal stability over 57 days. WB was prepared as described before and measured immediately after preparation (day 1) and then after storage in dark environment for 57 days under ambient conditions. FIG. 6 illustrates the use of a camera with ScanLater^TMOf boxes

Graph of the stability of the western blot results of the reader over time. The average band intensities of the two scans against background were analyzed and the results are shown in fig. 6. No degradation or signaling of WB was observed after 57 days of storageThe reduction in the number.

To investigate the effect of photobleaching, repeated reads were performed on WB using double serial dilutions of transferrin. Figure 7 shows a graph of TRF reagent lack of photobleaching after repeated scans of a single band on a Western Blot. For each scan, on the right is a bar graph showing integrated intensity ("integrated density"). The average intensity of the 250pg band for each scan was measured and the results are shown in FIG. 7. No systematic decrease in signal intensity was observed, indicating that there was no problem with photobleaching of the TRF agent.

Multiple detection of TRF has been reported with some success. The ability to detect two different proteins using Eu and Tb based probes has been demonstrated in biochemical assays using time-resolved fluorescence resonance energy transfer (TR-FRET) (Degorce et al (2009) curr. chem. genomics.3: 22-32; Bookout et al (2000) J. Agric. food chem.48(12): 5868-. In addition, there are reports of multiplex detection using Eu and Sm, and Eu, Tb and Sm (Bador et al (1987) Clin. chem.33(1): 48-51; Heinonen et al (1997) Clin. chem.43(7): 1142-1150). The analysis scheme of these systems uses a flash lamp with a monochromatic bandpass filter for excitation and multiple emission bandpass filters.

These systems of the prior art described above suffer from crosstalk because the emission from one of the lanthanides penetrates into the detection channel of the other lanthanides. In practice, good separation can be achieved in only one proportion due to the large number of emission peaks in the lanthanide spectra. For example, for Eu and Tb, there is minimal Eu signal in the Tb channel, but Tb crosstalk into the Tb channel may be as high as 10%. Eu and Sm enter the Eu channel reversely without Sm crosstalk, but significant (> 10%) Eu crosstalk enters the Sm channel. This limits the utility of these methods to only one truly sensitive channel, while the other is limited by the background signal from the second species. Fig. 8 and 9 show examples of these Eu, Tb and Sm dot blots. Fig. 8 shows Dot Blot results comparing cross-talk emission between detection channels of europium (Eu) and terbium (Tb) based probes. FIG. 9 shows Dot Blot results of cross-talk emission between detection channels of europium (Eu) and samarium (Sm) based probes.

As described elsewhere herein, one aspect of the invention relates to a novel method of using multiple long-life fluorescent dyes for TRF detection. We use a combination of spectral and temporal differences in fluorescence emission to enhance the ability to separate signals from the detection of multiple dyes. In some embodiments, this is simplified to practice using a combination of ruthenium (Ru) and europium (Eu) labels in a multiplex Western Blot detection scheme, but may also be applied to immunoassays, protein arrays, and other multiplex bioassays. Ru has been used as a dye for detecting proteins, DNA and other compounds, and takes advantage of its long life to create an assay system that rejects shorter-lived signals (Demas et al (1999) anal. chem.71(23): 793A-800A; Bertgren et al (1999) anal. biochem.276(2): 129-143; Ullmer et al (2012) Br.J. Pharmacol.167(7): 1448-1466). However, there has been no report on combining Ru and Eu or other lanthanides with very long lifetimes in a multiplex system.

The solution we developed takes advantage of both the temporal and wavelength domain differences between TRF dyes to reduce cross talk to less than 1%, more particularly to less than 0.01%. Time separation: the half-life was detected using a shorter time integration (2 microseconds) of

Microsecond Ru; detection of half-life as longer time integration (1000 microseconds)

Eu in microseconds. Spectral separation: ru was excited at 470nm and detected at 624 nm; eu is excited at 370nm and detected at 616 nm.

Fig. 10 shows the Western Blot results for GST dilution series, which compares the crosstalk emission between detection channels based on europium (Eu) and ruthenium (Ru) probes. These scans were obtained using a laser diode-excited cartridge.

FIG. 11 shows a plot of the average line scan through the channel on the Western Blot shown in FIG. 10; the line scan of the GST Eu-Eu cell is labeled 1101, the line scan of the GST Eu-Ru cell is labeled 1102, the line scan of the GST Ru-Ru cell is labeled 1103, and the line scan of the GST Ru-Eu cell is labeled 1104.

In that

These results are obtained using two different cartridges in the reader, but can be extended to

And

a single cartridge operating in the i3Multi-Mode micro plate Reader Detection Platform System (Molecular Devices, LLC, Sunnyvale, Calif.) of U.S.A.

Exemplary embodiments

Exemplary embodiments provided in accordance with the presently disclosed subject matter include, but are not limited to, the following:

1. a method for multiplex fluorescence detection, the method comprising: providing a sample comprising a first fluorescent label bound to a first analyte and a second fluorescent label bound to a second analyte, wherein the first fluorescent label comprises an up-converting phosphor (UCP) and the second fluorescent label comprises a non-UCP label; illuminating the first fluorescent label with a first excitation light of a first excitation wavelength, wherein the first fluorescent label emits a first detection signal of a first emission wavelength; illuminating the second fluorescent label with a second excitation light of a second excitation wavelength (which is different from the first excitation wavelength), wherein the second fluorescent label emits a second detection signal at the second emission wavelength; measuring an intensity of the first detection signal at a first measurement time, wherein the intensity of the first detection signal correlates to the amount of the first analyte in the sample;

stopping irradiating the second fluorescent marker; and measuring the intensity of a second detection signal at a second measurement time after ceasing to irradiate the second fluorescent marker, wherein the intensity of the second detection signal is correlated with the amount of the second analyte in the sample.

2. The method according to embodiment 1, comprising measuring the intensity of the first detection signal while illuminating the first fluorescent marker.

3. The method according to embodiment 1, comprising stopping the irradiation of the first fluorescent marker and measuring the intensity of the first detection signal after stopping the irradiation of the first fluorescent marker.

4. The method according to any one of the preceding embodiments, wherein the sample comprises a third fluorescent label bound to a third analyte, the third fluorescent label comprising a non-UCP label that is different from the second fluorescent label, and the method further comprises: illuminating a third fluorescent label with a third excitation light of a third excitation wavelength (which is different from the first excitation wavelength and the second excitation wavelength), wherein the third fluorescent label emits a third detection signal at a third emission wavelength; stopping the irradiation of the third fluorescent marker; and measuring the intensity of the third detection signal at a third measurement time after stopping the irradiation of the third fluorescent marker.

The method according to embodiment 4, comprising measuring the intensity of the first detection signal while illuminating the first fluorescent marker.

6. The method according to embodiment 4, comprising stopping the irradiation of the first fluorescent marker, and measuring the intensity of the first detection signal after stopping the irradiation of the first fluorescent marker.

7. The method according to any one of embodiments 4 to 6, wherein the second fluorescent label comprises a transition metal chelate and the third fluorescent label comprises a lanthanide chelate.

8. The method according to any one of embodiments 4 to 6, wherein: the second fluorescent label comprises a transition metal chelate selected from the group consisting of transition metal chelates of ruthenium (ru (ii)), osmium (os (ii)), and rhenium (re (i)); and the third fluorescent label comprises a lanthanide chelate selected from the group consisting of lanthanide chelates of samarium (sm) (iii), dysprosium (dy (iii), europium (eu (iii), and terbium (tb (iii)).

9. The method according to any one of embodiments 4 to 8, wherein: the first excitation wavelength is in a near infrared region, and the first emission wavelength is in a visible light region; and at least one of the second excitation wavelength and the third excitation wavelength is in the ultraviolet range.

10. The method according to any one of embodiments 4-9, wherein the UCP of the first fluorescent marker has a first fluorescence emission lifetime, the second fluorescent marker has a second fluorescence emission lifetime, the third fluorescent marker has a third fluorescence emission lifetime, and the first fluorescence emission lifetime is different from the second fluorescence emission lifetime and the third fluorescence emission lifetime.

11. The method according to any one of embodiments 4 to 9, wherein the UCP of the first fluorescent marker has a first fluorescence emission lifetime, the second fluorescent marker has a second fluorescence emission lifetime, the third fluorescent marker has a third fluorescence emission lifetime, and the second fluorescence emission lifetime is longer than the third fluorescence emission lifetime by a factor selected from the group consisting of at least 5-fold, at least 50-fold, at least 100-fold, at least 500-fold, and at least 1000-fold.

12. The method according to any one of embodiments 4 to 9, wherein the UCP of the first fluorescent marker has a first fluorescence emission lifetime and the second fluorescent marker has a second fluorescence emission lifetime, and comprising at least one of: at least one of the second fluorescence emission lifetime and the third emission lifetime is in a range of 0.1 μ s to 10 μ s; at least one of the second fluorescence emission lifetime and the third emission lifetime is in a range of 100 μ s to 1 ms.

13. The method according to any one of embodiments 4 to 12, wherein the stokes shift of at least one of the second fluorescent marker and the third fluorescent marker is selected from the group consisting of: stokes shift greater than 20 nm; stokes shift greater than 100 nm; stokes shift greater than 250 nm; and a stokes shift in the range of about 250nm to about 350 nm.

14. The method according to any one of embodiments 4-13, wherein the second emission wavelength is different from the first emission wavelength.

15. The method according to any one of embodiments 4-14, wherein the second measurement time is different from the first measurement time.

16. The method according to any one of embodiments 4-15, wherein the third emission wavelength is different from the first emission wavelength and the second emission wavelength.

17. The method according to any one of embodiments 4-16, wherein the third measurement time is different from the first measurement time and the second measurement time.

18. The method according to any one of the preceding embodiments, wherein providing the sample comprises: contacting the sample with: a first antibody that specifically binds to a first analyte; a second antibody that specifically binds to a second analyte; a third antibody that specifically binds to a third analyte; a first fluorescent antibody conjugate that specifically binds to a first antibody, wherein the first fluorescent antibody conjugate comprises a first fluorescent label; and a second fluorescent antibody conjugate that specifically binds to a second antibody, wherein the second fluorescent antibody conjugate comprises a second fluorescent label; a third fluorescent antibody conjugate that specifically binds to a third antibody, wherein the third fluorescent antibody conjugate comprises a third fluorescent label; and incubating the sample under conditions and for a time sufficient for the antibody and antibody conjugate to form an immune complex.

19. The method according to any one of embodiments 4 to 17, wherein providing the sample comprises contacting the sample with a first antibody that specifically binds to the first analyte, a second antibody that specifically binds to the second analyte, and a third antibody that specifically binds to the third analyte, wherein at least one of the first fluorescent label, the second fluorescent label, and the third fluorescent label is directly attached to the respective first antibody, the second antibody, or the third antibody.

20. The method according to any one of the preceding embodiments, wherein the first, second and third analytes comprise proteins or membrane-bound proteins.

21. The method according to any one of the preceding embodiments, wherein at least one of the first, second and third analytes is a reference protein and at least another one of the first, second and third analytes is an unknown protein, and further comprising: the detection signal obtained from the unknown protein is normalized to the detection signal obtained from the reference protein.

22. A method according to any one of the preceding embodiments, wherein at least one of the first, second and third analytes is an unmodified protein and at least another one of the first, second and third analytes is a modified or phosphorylated form of the protein, and further comprising: the ratio of modified or phosphorylated form of protein to unmodified protein is calculated based on the measured intensity of the detection signal obtained from unmodified protein, and modified or phosphorylated form of protein.

23. The method of any of the preceding embodiments, comprising directing at least two of the first detection signal, the second detection signal, and the third detection signal through a common emission filter.

24. The method according to any one of the preceding embodiments, wherein the UCP comprises a lanthanide-doped inorganic compound or a transition metal-doped inorganic compound exhibiting anti-stokes shift.

25. The method of embodiment 24, wherein the inorganic compound comprises an element selected from the group consisting of erbium (Er)³⁺) Thulium (Tm)³⁺) Holmium (Ho)³⁺) Praseodymium (Pr)³⁺) Neodymium (Nd)³⁺) Dysprosium (Dy)³⁺) Ytterbium (Yb)³⁺) Samarium (Sm)³⁺) And combinations of two or more of the foregoing.

26. The method according to embodiment 24 or 25, wherein the inorganic compound is selected from the group consisting of halides, oxides and oxysulfides.

27. The method according to any one of the preceding embodiments, wherein the second fluorescent label comprises a transition metal chelate or a lanthanide chelate.

28. The method according to any one of the preceding embodiments, wherein the second fluorescent label comprises a transition metal chelate selected from the group consisting of transition metal chelates of ruthenium (ru (ii)), osmium (os (ii)), and rhenium (re (i)).

29. The method according to any one of the preceding embodiments, wherein the second fluorescent label comprises a lanthanide chelate selected from the group consisting of lanthanide chelates of lanthanum (sm (iii), dysprosium (dy (iii), europium (eu (iii)), and terbium (tb (iii)).

30. The method according to any one of the preceding embodiments, wherein the first excitation wavelength is in the near infrared range and the first emission wavelength is in the visible range.

31. The method according to any of the preceding embodiments, wherein the second excitation wavelength is in the ultraviolet range and the second emission wavelength is longer than the second excitation wavelength.

32. The method according to any one of the preceding embodiments, wherein the UCP of the first fluorescent marker has a first fluorescence emission lifetime, the second fluorescent marker has a second fluorescence emission lifetime, and the first fluorescence emission lifetime is different from the second fluorescence emission lifetime.

33. The method according to any one of the preceding embodiments, wherein the UCP of the first fluorescent marker has a first fluorescence emission lifetime and the second fluorescent marker has a second fluorescence emission lifetime, and comprising at least one of: the second fluorescence emission lifetime is in the range of 0.1 μ s to 10 μ s; the second fluorescence emission lifetime is in the range of 100 mus to 1 ms.

34. The method according to any one of the preceding embodiments, wherein the stokes shift of the second fluorescent marker is selected from the group consisting of: stokes shift greater than 20 nm; stokes shift greater than 100 nm; stokes shift greater than 250 nm; and a stokes shift in the range of about 250nm to about 350 nm.

35. The method of any one of the preceding embodiments, wherein the second emission wavelength is different from the first emission wavelength.

36. The method according to any one of the preceding embodiments, wherein the second measurement time is different from the first measurement time.

37. The method according to any one of the preceding embodiments, wherein providing the sample comprises: contacting the sample with: a first antibody that specifically binds to a first analyte; a second antibody that specifically binds to a second analyte; a first fluorescent antibody conjugate that specifically binds to a first antibody, wherein the first fluorescent antibody conjugate comprises a first fluorescent label; a second fluorescent antibody conjugate that specifically binds to a second antibody, wherein the second fluorescent antibody conjugate comprises a second fluorescent label; and incubating the sample under conditions and for a time sufficient for the antibody and antibody conjugate to form an immune complex.

38. The method according to any one of embodiments 1 to 36, wherein providing the sample comprises: contacting the sample with a first antibody that specifically binds to a first analyte, and a second antibody that specifically binds to a second analyte, wherein the first fluorescent label is directly attached to the first antibody, or the second fluorescent label is directly attached to the second antibody, or the first fluorescent label is directly attached to the first antibody and the second fluorescent label is directly attached to the second antibody.

39. The method according to any one of the preceding embodiments, wherein the first analyte and the second analyte comprise proteins or membrane-bound proteins.

40. The method according to any one of the preceding embodiments, wherein one of the first and second analytes is a reference protein and the other of the first and second analytes is an unknown protein, and further comprising: the second detection signal is normalized to the first detection signal or the first detection signal is normalized to the second detection signal.

41. A method according to any one of the preceding embodiments, wherein one of the first and second analytes is an unmodified protein and the other of the first and second analytes is a modified or phosphorylated form of the protein, and further comprising: the ratio of the modified or phosphorylated form of the protein to the unmodified protein is calculated based on the measured intensities of the first and second detection signals.

42. The method according to any of the preceding embodiments, comprising directing the first detection signal and the second detection signal through a common emission filter or through different emission filters.

43. The method according to any one of the preceding embodiments, wherein the irradiation of the first fluorescent marker and the irradiation of the second fluorescent marker are performed simultaneously or sequentially.

44. The method according to any one of the preceding embodiments, wherein providing a sample comprises providing a plurality of samples, and further comprising: performing a step of irradiating the first fluorescent marker, a step of irradiating the second fluorescent marker, a step of measuring the intensity of the first detection signal, and a step of measuring the intensity of the second detection signal for each sample, thereby performing multiplex fluorescence detection for each sample.

45. The method of embodiment 44, wherein performing multiple fluorescence detections on each sample comprises optically aligning each sample with a light source and a light detector, wherein the light source is configured to generate first excitation light and second excitation light, and the light detector is configured to measure first detection signals and second detection signals.

46. The method according to embodiment 45, wherein: optically aligning each sample with the light source and the light detector comprises: optically aligning each sample with the cartridge; the cassette is removably mounted in the device housing of the fluorescence detection device such that the cassette is in optical and/or electrical communication with the fluorescence detection device; the cartridge encloses excitation optics that define an optical path from the light source to the aligned sample, or the cartridge encloses emission optics that define an optical path from the aligned sample to the light detector, or the cartridge encloses both; disposing the light source in a cartridge or in a device housing; and the light detector is disposed in the cartridge or in the device housing.

47. The method according to any one of embodiments 44 to 46, wherein the samples are provided in separate wells of a multi-well plate, or in separate blots of a membrane, respectively.

48. A fluorescence detection device configured for performing at least the illuminating and measuring steps in the method of any of the preceding embodiments, the fluorescence detection device comprising: a light source configured to generate first excitation light and second excitation light; and a photodetector configured to measure the first detection signal and the second detection signal.

49. The fluorescence detection device according to embodiment 48, comprising at least one of: a light source including a first light source configured to generate first excitation light, and a second light source configured to generate second excitation light; a light detector including a first light detector configured to measure a first detection signal, and a second light detector configured to measure a second detection signal.

50. The fluorescence detection device according to embodiment 49, further comprising a third light source configured to generate third excitation light, and a third light detector configured to measure a third detection signal.

51. A fluorescence detection device, comprising: a sample support configured to support a sample, the sample comprising a first fluorescent label bound to a first analyte and a second fluorescent label bound to a second analyte, wherein the first fluorescent label comprises an up-converting phosphor (UCP) and the second fluorescent label comprises a non-UCP label; a light source configured to generate first excitation light of a first excitation wavelength and second excitation light of a second excitation wavelength (which is different from the first excitation wavelength); a photodetector configured to measure a first detection signal of a first emission wavelength emitted from the sample in response to excitation by the first excitation light, and a second detection signal of a second emission wavelength emitted from the sample in response to excitation by the second excitation light; and a computing device configured to: controlling the light source to generate first excitation light and second excitation light, respectively, for a predetermined excitation time and for a predetermined time; and controlling the light detector to measure the first detection signal at a first measurement time and to measure the second detection signal at a second measurement time.

52. The fluorescence detection apparatus according to embodiment 51, wherein the computing device is configured to receive an electrical output from the photodetector corresponding to the measurement of the first detection signal and the second detection signal, and correlate the measurement with the amount of the first analyte in the sample and the amount of the second analyte in the sample.

53. The fluorescence detection device according to embodiment 51, wherein the computing apparatus is configured to control the light source to stop generating the second excitation light and the light detector to measure the second detection signal after stopping generating the second excitation light.

54. The fluorescence detection device according to any one of embodiments 48-53, wherein: the sample comprises a third fluorescent label bound to a third analyte, the third fluorescent label comprising a non-UCP label different from the second fluorescent label; the light source is configured to generate third excitation light at a third excitation wavelength (which is different from the first excitation wavelength and the second excitation wavelength); the light detector is configured to measure a third detection signal at a third emission wavelength; and the computing device is configured to: controlling the light source to generate third excitation light for a predetermined excitation time; and controlling the light detector to measure a third detection signal at a third measurement time.

55. The fluorescence detection device according to embodiment 54, wherein the computing apparatus is configured to control the light source to stop generating the third excitation light, and to control the light detector to measure the third detection signal at a third measurement time different from the first measurement time and the second measurement time after the generation of the third excitation light is stopped.

56. The fluorescence detection device according to any one of embodiments 48 to 55, comprising a device housing, a cartridge removably mounted in the device housing, excitation optics configured to define an optical path from the light source to the sample, and emission optics configured to define an optical path from the sample to the light detector, wherein: disposing a light source in a cartridge or in a device housing; disposing a light detector in a cartridge or in a device housing; and disposing the computing device in the apparatus housing.

57. The fluorescence detection apparatus of embodiment 56, wherein at least one of the excitation optics and the emission optics are disposed in a cartridge.

58. The fluorescence detection device according to any one of embodiments 48 to 57, wherein the sample support comprises or is configured for supporting a multi-well plate or a multi-blotting membrane.

It will be understood that one or more of the processes, sub-processes and process steps described herein may be performed on one or more electronic or digitally controlled devices by hardware, firmware, software, or a combination of two or more of the foregoing. The software may reside in a software memory (not shown) of a suitable electronic processing component or system, such as the computing device 236 schematically depicted in fig. 2. The software memory may include an ordered listing of executable instructions for implementing logical functions (i.e., "logic" that may be implemented in digital form (e.g., digital circuitry or source code) or in analog form (e.g., an analog power supply such as an analog electrical, acoustic, or video signal)). The instructions may be executed within a processing module that includes, for example, one or more microprocessors, general-purpose processors, combinations of processors, Digital Signal Processors (DSPs), or Application Specific Integrated Circuits (ASICs). Further, the schematic diagrams depict a logical division of functions having a physical (hardware and/or software) implementation that is not limited by the physical layout of the architecture or functions. Examples of the systems described herein may be implemented in various configurations and may operate as hardware/software components in a single hardware/software unit or as separate hardware/software units.

Executable instructions may be implemented as a computer program product having instructions stored therein that, when executed by a processing module of an electronic system (e.g., computing device 236 in fig. 2), direct the electronic system to perform the instructions. A computer program product may optionally be embodied in any non-transitory computer-readable storage medium for use by or in connection with an instruction execution system, apparatus, or device, such as an electronic computer-based system, processor-containing system, or other system that may selectively fetch the instructions from the instruction execution system, apparatus, or device and execute the instructions. In the context of this disclosure, a computer-readable storage medium is any non-transitory apparatus that can store a program for use by or in connection with an instruction execution system, apparatus, or device. The non-transitory computer readable storage medium can alternatively be, for example, an electronic, magnetic, optical, electromagnetic, infrared, or semiconductor system, apparatus, or device. A non-exhaustive list of more specific examples of the non-transitory computer-readable medium includes: an electrical connection having one or more wires (electrical); portable computer diskette (magnetic); random access memory (electronic); read-only memory (electronic); erasable programmable read-only memory such as, for example, flash (electronic); optical disk storage such as, for example, CD-ROM, CD-R, CD-RW (optical); and digital versatile disc storage, i.e., DVD (optical). Note that the non-transitory computer-readable storage medium could even be paper or another suitable medium upon which the program is printed, as the program can be electronically captured, via, for instance, optical scanning of the paper or other medium, then compiled, interpreted, or otherwise processed in a suitable manner, if necessary, and then stored in a computer or machine memory.

It will also be understood that the term "signal communication" as used herein means that two or more systems, devices, components, modules or sub-modules are capable of communicating with each other via signals propagating on some type of signal path. A signal may be a communication, electrical, data, or energy signal, wherein the signal may convey information, electricity, or energy from a first system, device, component, module, or sub-module to a second system, device, component, module, or sub-module along a signal path between the first and second systems, devices, components, modules, or sub-modules. The signal path may include a physical connection, an electrical connection, a magnetic connection, an electromagnetic connection, an electrochemical connection, an optical connection, a wired connection, or a wireless connection. The signal path may also include additional systems, devices, components, modules, or sub-modules between the first and second systems, devices, components, modules, or sub-modules.

More generally, terms such as "communicate" and "with …" (e.g., "a first component communicates" and "with a second component communicates") are used herein to indicate a structural, functional, mechanical, electrical, signaling, optical, magnetic, electromagnetic, ionic, or fluidic relationship between two or more components or elements. Thus, the fact that one component is considered to be in communication with a second component is not intended to exclude the possibility that additional components may be present between and/or operatively associated or engaged with the first and second components.

It will be understood that various aspects or details of the invention may be changed without departing from the scope of the invention. Furthermore, the foregoing description is for the purpose of illustration only, and not for the purpose of limitation, i.e., the invention is defined by the claims.

Claims (16)

1. A method for multiplex fluorescence detection, the method comprising:

providing a sample comprising a first fluorescent label bound to a first analyte and a second fluorescent label bound to a second analyte;

wherein the first fluorescent label is selected from the group consisting of transition metal chelates of ruthenium Ru (II), osmium Os (II), and rhenium Re (I), and the second fluorescent label is selected from the group consisting of lanthanide chelates of samarium Sm (III), dysprosium Dy (III), europium Eu (III), and terbium Tb (III);

illuminating the first fluorescent marker with a first excitation light having a first excitation wavelength, wherein the first fluorescent marker emits a first detection signal having a first emission wavelength;

illuminating the second fluorescent marker with a second excitation light having a second excitation wavelength different from the first excitation wavelength, wherein the second fluorescent marker emits a second detection signal having a second emission wavelength;

measuring the intensity of the first detection signal at a first measurement time, wherein the intensity of the first detection signal is correlated with the amount of the first analyte in the sample;

stopping irradiation of the second fluorescent marker; and

measuring the intensity of the second detection signal at a second measurement time after stopping the irradiation of the second fluorescent marker, wherein the intensity of the second detection signal is correlated with the amount of the second analyte in the sample.

2. The method of claim 1, comprising measuring the intensity of the first detection signal while illuminating the first fluorescent marker.

3. The method of claim 1, comprising stopping the irradiation of the first fluorescent marker, and measuring the intensity of the first detection signal after stopping the irradiation of the first fluorescent marker.

4. The method of claim 1, wherein the first excitation wavelength is in the near infrared range and the first emission wavelength is in the visible range.

5. The method of claim 1, wherein the second excitation wavelength is in the ultraviolet range and the second emission wavelength is longer than the second excitation wavelength.

6. The method of claim 1, wherein the sample comprises a third fluorescent label bound to a third analyte, and the method further comprises:

illuminating the third fluorescent marker with a third excitation light having a third excitation wavelength different from the first excitation wavelength and the second excitation wavelength, wherein the third fluorescent marker emits a third detection signal having a third emission wavelength;

stopping irradiating the third fluorescent marker; and

measuring the intensity of the third detection signal at a third measurement time after ceasing to irradiate the third fluorescent marker.

7. The method of claim 6, comprising at least one of:

wherein the third emission wavelength is different from the first emission wavelength and the second emission wavelength;

wherein the third measurement time is different from the first measurement time and the second measurement time.

8. The method of claim 1, wherein the first fluorescent marker has a first fluorescent emission lifetime, the second fluorescent marker has a second fluorescent emission lifetime, and the second fluorescent emission lifetime is selected from the group consisting of:

the second fluorescence emission lifetime is different from the first fluorescence emission lifetime;

the second fluorescence emission lifetime is longer than the first fluorescence emission lifetime;

the second fluorescence emission lifetime is at least 5 times longer than the first fluorescence emission lifetime;

the second fluorescence emission lifetime is at least 100 times longer than the first fluorescence emission lifetime;

the second fluorescence emission lifetime is at least 1,000 times longer than the first fluorescence emission lifetime;

the second fluorescence emission lifetime is in the range of 0.1 μ s to 10 μ s; and

the second fluorescence emission lifetime is in the range of 100 μ s to 1 ms.

9. The method of claim 1, wherein the stokes shift of at least one of the first fluorescent marker and the second fluorescent marker is selected from the group consisting of: stokes shift greater than 20 nm; stokes shift greater than 100 nm; stokes shift greater than 250 nm; and a stokes shift in the range of 250nm to 350 nm.

10. The method of claim 1, comprising at least one of:

wherein the second emission wavelength is different from the first emission wavelength;

wherein the second measurement time is different from the first measurement time.

11. The method of claim 1, wherein the first and second analytes comprise proteins or membrane-bound proteins, and further comprising at least one of:

wherein one of the first and second analytes is a reference protein and the other of the first and second analytes is an unknown protein, and further comprising normalizing the second detection signal to the first detection signal or normalizing the first detection signal to the second detection signal;

wherein one of the first and second analytes is an unmodified protein and the other of the first and second analytes is a modified or phosphorylated form of the protein, and further comprising calculating a ratio of the modified or phosphorylated form of the protein to the unmodified protein based on the measured intensities of the first and second detection signals.

12. The method of claim 1, wherein illuminating the first fluorescent marker and illuminating the second fluorescent marker are performed simultaneously or sequentially.

13. The method of claim 1, comprising optically aligning the sample with a cartridge, wherein:

removably mounting the cartridge in a device housing of a fluorescence detection device such that the cartridge is in optical and/or electrical communication with the fluorescence detection device;

the cartridge enclosing excitation optics defining an optical path from a light source to an aligned sample, or enclosing emission optics defining an optical path from the aligned sample to the light detector, or enclosing the aforementioned excitation and emission optics;

disposing the light source in the cartridge or in the device housing; and

disposing the light detector in the cartridge or in the device housing.

14. A fluorescence detection device configured to perform a method comprising:

illuminating a first fluorescent label bound to a first analyte of the sample with a first excitation light having a first excitation wavelength, wherein the first fluorescent label emits a first detection signal having a first emission wavelength, wherein the first fluorescent label is selected from the group consisting of transition metal chelates of ruthenium ru (ii), osmium os (ii), and rhenium re (i);

illuminating a second fluorescent label bound to a second analyte of the sample with a second excitation light having a second excitation wavelength different from the first excitation wavelength, wherein the second fluorescent label emits a second detection signal having a second emission wavelength, wherein the second fluorescent label is selected from the group consisting of lanthanide chelates of samarium sm (iii), dysprosium dy (iii), europium eu (iii), and terbium tb (iii); and

measuring the intensity of the first detection signal at a first measurement time, wherein the intensity of the first detection signal is correlated with the amount of the first analyte in the sample;

wherein the fluorescence detection device comprises:

a light source configured to generate the first excitation light and the second excitation light; and

a photodetector configured to measure the first detection signal and the second detection signal.

15. A fluorescence detection device, comprising:

a sample support configured to support a sample comprising a first fluorescent label bound to a first analyte and a second fluorescent label bound to a second analyte;

wherein the first fluorescent label is selected from the group consisting of transition metal chelates of ruthenium Ru (II), osmium Os (II), and rhenium Re (I), and the second fluorescent label is selected from the group consisting of lanthanide chelates of samarium Sm (III), dysprosium Dy (III), europium Eu (III), and terbium Tb (III);

a light source configured to generate first excitation light having a first excitation wavelength and second excitation light having a second excitation wavelength different from the first excitation wavelength;

a photodetector configured to measure a first detection signal having a first emission wavelength emitted from the sample in response to excitation by the first excitation light and a second detection signal having a second emission wavelength emitted from the sample in response to excitation by the second excitation light; and

a computing device configured to:

controlling the light source to generate the first excitation light and the second excitation light, respectively, at predetermined excitation times for predetermined times; and

controlling the light detector to measure the first detection signal at a first measurement time and to measure the second detection signal at a second measurement time.

16. The fluorescence detection device of claim 15, comprising a device housing, a cartridge removably mounted in the device housing, excitation optics configured to define an optical path from the light source to the sample, and emission optics configured to define an optical path from the sample to the light detector, wherein:

disposing the light source in the cartridge or in the device housing;

disposing the light detector in the cartridge or in the device housing; and is

Disposing at least a portion of the excitation optics, or at least a portion of the emission optics, or at least a portion of the excitation optics and at least a portion of the emission optics in the cartridge or in the device housing.

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